Abstract
A mutant Chinese hamster ovary cell line, glyB, that required exogenous glycine for survival and growth was reported previously (Kao, F., Chasin, L., and Puck, T. T. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 1284-1291). We now report that the defect in glyB cells causative of this phenotype is a point mutation in an inner mitochondrial membrane protein required for transport of folates into mitochondria. The CHO mitochondrial folate transporter (mft) was sequenced and compared with that from glyB cells. The hamster sequence was nearly identical to that of the recently reported human mitochondrial folate transporter. The corresponding cDNA from glyB cells contained a single nucleotide change that introduced a glutamate in place of the glycine in wild-type hamster MFT at codon 192 in a predicted transmembrane domain. Transfection of the wild-type hamster cDNA into glyB cells allowed cell survival in the absence of glycine and the accumulation of folates in mitochondria, whereas transfection of the Glu-192 cDNA did not. Genomic sequence analysis and fluorescence in situ hybridization demonstrated a single mutated allele of the mft gene in glyB cells, whereas there were two alleles in CHO cells. We conclude that we have defined the cause of the glyB auxotrophy and that the glyB mft mutation identified a region of this mitochondrial folate carrier vital to its transport function.
Highlights
It remained possible that the complementation observed could have been through a compensatory or nonspecific mechanism, and direct evidence that the CHO mitochondrial folate transporter (MFT) was defective in glyB cells was needed to conclude that this protein is the endogenous mitochondrial transporter that facilitates the entry of folates into the mitochondrial matrix
The Mitochondrial Folate Transporter Is Expressed in glyB Cells—Earlier studies by Kao et al [1] and Chasin et al [14] establish four complementation groups among CHO mutants that were auxotrophic for glycine
We report that the homologous hamster protein is mutated in glyB cells and that the single point mutation distinguishing the hamster MFT proteins in CHO and glyB cells explains the mitochondrial folate transport defect in glyB cells
Summary
Materials—CHO-K1 cells were obtained from ATCC. glyB cells, a CHO-K1 derivative, were generously provided by Prof. A 313-nt PCR-amplified fragment from the 5Ј end of CHO mft cDNA was 32Plabeled by random priming and added to the membranes of duplicate nitrocellulose lifts in a hybridization mixture at 1 ϫ 106 cpm/ml This PCR product was cloned and sequenced prior to use, and its identity as an upstream fragment of the hamster mft was deduced from homology with the human mft. Two fragments of CHO genomic DNA were PCR-amplified and directly labeled with the Spectrum Orange fluorophore using a nick translation kit (Vysis) These fragments corresponded to the regions of the hamster mft gene spanning predicted exons 2– 4 including the two intervening introns (5.7 kb) and the region spanning predicted exons 6 and 7 including the intervening intron (1.3 kb). Representative images were documented using a Cytovision image analysis system (Applied Imaging)
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